JP4934052B2 - New microfluidic sample holder - Google Patents

Abstract

A novel microfluidic sample holder has at least one sample receiving compartment for a sample fluid, at least one distributor channel which is linked with at least one sample receiving compartment, at least one distributor channel extending from every sample receiving compartment, at least one reaction chamber to which optionally one inlet channel branched off from the at least one distributor channel leads, and at least one vent opening for every reaction chamber. The sample holders are mainly used in microbiological diagnostics, immunology, PCR, clinical chemistry, microanalytics and/or the inspection of active substances. Methods for analyzing a sample substance using the sample holder and to kits including the sample holder as also included.

Description

The present invention relates to a novel microfluidic sample holder and relates to at least one fluid sample sample receiving chamber, at least one distributor channel connected to the sample receiving chamber, and at least one distributor extending from each material receiving chamber. A microfluidic sample holder comprising a flow channel, where appropriate, at least one reaction chamber open with an inlet flow channel branched by at least one distributor flow channel, and at least one vent for each reaction chamber . This sample holder is mainly useful for use in microbiological diagnostics, immunology, PCR methods, clinical chemistry, microanalysis and / or active substance testing. The invention further relates to a sample material analysis method using a sample holder and a kit including the sample holder.

The great progress in biodevice development has also opened a new dimension in medical diagnostics. In light of the increasing severity of the problem of paying public health costs, the possibility of cost reduction is considered particularly important here. Advances in science and technology have suggested a number of ways how diagnostic problems can be improved with the help of multi-parameter tests in the last few years. The greatest success here is the development of the so-called biodevice field, in particular the DNA chip field. At the same time, other test formats such as bead technology and microfluidic systems have been developed.
Microfluidics is usually understood to handle and manage trace amounts of fluid (eg, microliters, nanoliters or even picoliters). Various methods are used for the intended movement of the fluid.
Electrokinetic pressure capillary phenomenon These can be applied separately or in combination. In this case, the dynamic current can be obtained by applying a voltage to the flow path. Transport of charged molecules occurs by a phenomenon known as electroosmosis and electrophoresis. In contrast, it is also possible to transport uncharged molecules and, for example, cells to be moved, by pressurization (eg with a micropump). Passive methods are increasingly used with these activation methods. In this case, the fluid can be transported in a desired form using capillary force. An important advantage of this technique is that it can be managed without additional drive mechanisms, so that the entire system can be greatly simplified.
As seen in global terms, most solutions concentrate on “active elements” for fluid transport. The structure required in this case is overwhelmingly created by laser ablation, hot forging or injection molding. This often limits the possibility of structuring. The first solution for passive transport of fluids already exists in Germany. In these cases, molded products are currently produced by microinjection molding, and the energy required for fluid transport is currently provided by surface hydrophilization by plasma treatment. The disadvantages of this technique are that the hydrophilized thin layer tends to become anisotropic on the surface (aging due to hydrophobic recovery) and has a relatively high sensitivity to chemicals and solvents. An alternative to photolithography-based methods is provided. Here, the structure is created by photopolymerization of an acrylate ester with the aid of an optical mask. A copolymer having the desired surface properties can be prepared by adding an appropriate crosslinkable organic substance. Furthermore, this method cannot be performed on the other side, or the generation of three-dimensional structures that can only be performed at unacceptable costs can be omitted.
This sample holder which uses only capillary forces for sample fluid transport is known, for example, from WO 99/46045. Included here are plastic chips made using microinjection molding and then modified (hydrophilized) by plasma treatment or grafting of the surface. These methods are expensive and have a series of disadvantages.
1. Surface modification cannot be maintained for a sufficient period due to hydrophobic recovery, and uniformity cannot be controlled in the three-dimensional direction.
2. Particularly in the case of aggregate testing, substances that are at risk of clogging capillaries due to excessive hyperhydrophilicity result in unwanted capillaryization returning to the inlet and venting capillaries. As a result, the sample holder cannot be used.
3. Capillaries can be easily formed between the recess wall and the cover of the injection point into the test recess (especially with poor sealing or with hydrophilic adhesive), so that the fluid can no longer be vented into the adjacent recess, so it is no longer filled In order not to be able to do so, it directly flows into and fills the vent structure, so that the recess is not filled or is incompletely filled. Furthermore, in this case, the fluid capillaries beyond the outer edge of the sample holder into a further vent structure belonging to another test.
4). However, the free surface energy of the sample holder, which aims to ensure fluid transport to the test recess, is very sensitive to the surfactant in the fluid, since the above drawbacks occur here strongly. As a result, non-ionic surfactants are essential in many diagnostic assays (immunoassay, DNA analysis, clinical chemistry), thus eliminating many potential applications.
5. The above sample holder can only be used for one-step assays.
Microfluidic chips and / or sample holders offer the potential to improve sample throughput while at the same time greatly reducing diagnostic methods. Based on this reduction, faster reaction, higher sensitivity and better control can be obtained in the whole procedure compared to the conventional method. Therefore, the development of a reliably functioning microfluidic chip or sample holder is a decisive event in the middle of an innovative miniaturized diagnostic system.
Microfluidic chips or sample holders contain three-dimensional elements with very different dimensions. Thus, for example, at the transition between the capillary and the “reaction cavity”, a laminar flow of fluid needs to be directed to the bottom of the vessel to completely fill the vessel. In addition, there is a possibility of flow in the other direction due to the capillary action occurring especially at the reaction vessel cover and side walls. As a result, an uncontrollable chaotic flow is expected in this transition. Therefore, a microfluidic structure that ensures the complete filling of the reaction cavity under the worst conditions is essential but still does not exist.

In this invention, a new sample holder, a sample material analysis method using this new sample holder, and the disadvantages existing in the prior art are solved, in particular, the injection kinetics is improved, the low sensitivity is relaxed, and the simple and cost-effective method. We address the objective of providing a kit that includes a one-step assay, or a new sample holder that helps provide a specific and sensitive multi-step assay sufficient to ensure rapid quantitative identification of, for example, sample material.
This object is achieved by the invention with independent claim characteristics. The beneficial development of the invention has the features of the subclaims. All wordings of the claims are incorporated herein by reference.
Improved microfluidic samples by a series of methods involving structure shapes suitable for applications in fluid samples and test assemblies, the placement of specific structural elements, the use of surface free energy gradients in the vertical direction, and nonionic surfactants It is possible to provide a holder. Particularly important points in this case are as follows.
Investigation of the influence of the new design specific capacity (height of the structure) of the distribution channel and the ventilation channel Investigation of the effect of the capillary plug structure on the flow behavior of the fluid surface energy Statistical analysis of the injection time Each method will be described in more detail. The steps need not necessarily be performed in a particular order, and the summary method may further include unspecified steps.
At least one fluid sample sample receiving chamber, at least one distributor channel connected to the one sample receiving chamber, at least one distributor channel extending from each material receiving chamber, and at least one, if appropriate. A sample holder is provided having at least one reaction chamber open with an inlet channel branched by one distributor channel, and at least one vent for each reaction chamber. The sample holder is at least partially added with a hydrophobic design between the sample receiving chamber, the distributor channel, the reaction chamber, the inlet channel and / or the vent channel, where appropriate. Has a structure. On the one hand it tries to let the air displaced by the inflowing fluid escape (sample receiving chamber → distributor → reaction chamber), and on the other hand it cancels the capillarity or strongly delays (capillary plug) the structure is perfect if appropriate It has a hydrophobic design. The size of these structures is preferably relatively small, for example the cross section of each additional structure is from about 10 μm to about 300 μm, preferably from about 50 μm to about 200 μm, especially from about 100 μm to about 150 μm. It is important to point out in the text that in any case these structures are not so small as to be blocked when covered with cover elements as will be described later. Seal the sample holder (after reagent introduction if appropriate) for capillary action sufficient for the sample fluid in the microfluidic sample holder to be transported passively, but the sample holder is probably covered when covered with a cover element It is important that it is not shielded in use, for example with an adhesive.

BEST MODE FOR CARRYING OUT THE INVENTION

In a preferred embodiment, the additional structure is preferably a substantially semi-circular recess located opposite the distributor flow path. At least one additional capillary preferably extends from the semicircular recess, and the additional capillary is designed to be acute, preferably at an angle of 90 degrees or more, and / or bend zigzag. This additional capillary that can be placed on the distributor wall retards or stops fluid flow due to its capillary structure. In a further preferred embodiment, the additional capillary is followed by at least one additional element with a change in structure depth that enhances the effect substantially sharp. If at least one additional element extends from an element having a substantially sharp structure depth change, it is advantageous for this additional capillary to open directly or through an adjacent structure into the terminal recess having a valve function. The adjacent structure may be, for example, a common main ventilation path opened by at least one ventilation hole. If this additional capillary is sealed, for example with the aid of foil, pressure replenishment does not occur, so that the (total) capillary forces may cancel each other. Upon opening the seal (eg, piercing the foil or using a focused laser), the structure achieves its intended purpose, ie, injection begins or continues with the aid of capillary forces. The vent structure may also continue from various structures, such as a sample receiving chamber, a distributor channel, a reaction chamber, an inlet channel, a distributor channel and / or an inlet channel connected to additional structures, etc. In this case, the vent structure and / or the vent are initially closed. In this case, the open vent structure of the first test recess (eg the first reaction chamber) ends at that side. When sample material is added thereto, the first recess is injected so that the first stage reaction proceeds. Thereafter, the vent system of the second test recess (eg, the second reaction chamber), preferably having a smaller volume, is opened and the changed sample material is injected from the first recess. The second stage reaction proceeds.
In a further preferred embodiment, the inlet channel is flush with the vent in the upper region. This inlet channel is preferably of a substantially hydrophobic design in this region. The lower region of the inlet channel, i.e., the region below the vent surface, is preferably of a substantially hydrophilic design. As an alternative to this, only the inlet channel can be processed with a more hydrophilic material (compared to the material used in the upper region). In WO99 / 46045, sample distribution is performed by a distributor flow path that starts from a sample addition point and branches into a sample recess (for example, a reaction chamber) at an inlet flow path. While this sample distribution system is well known, for the reasons described above, this system is not suitable for ensuring proper injection kinetics. Thus, the inlet channel and / or the distribution channel can preferably also continue individually from the sample receiving chamber. In addition, the distributor channel connected to the sample receiving chamber is preferably of a serpentine design and can be directly connected to the sample receiving chamber (ie without intervening inlet channels). Of course, the distributor channel function is assumed or supplemented by an inlet channel that may be present there, such that serpentine shapes on the distributor channel and / or inlet channel are included in the invention. In addition, a number of inlets, distributor channels, where appropriate inlet channels, reaction chambers and / or additional structures can be arranged around the sample receiving chamber, preferably parallel thereto. This arrangement consists, for example, of a “jellyfish shape”, the function of the “jellyfish head” being assumed by the sample receiving chamber and the function of the “jellyfish tentacle” being assumed by the distributor channel and / or the inlet channel. According to the invention, the sample receiving chamber is formed in the center of a circle, ellipse or elongated structure (so-called “arthropod structure”), the distributor channel and / or the inlet channel (and / or additional structures) there Keep away from. Arrangements that allow two-step assays or multi-step assays can be correspondingly arranged.
According to an advantageous result of the invention, the vertical length of the reaction chamber is from about 500 μm to about 3 mm, preferably from about 1 mm to about 2.5 mm, in particular from about 1.5 mm to about 2 mm. The side length of the reaction chamber is on average about 300 μm to about 1 mm, preferably about 500 μm to about 750 μm, especially about 500 μm to about 600 μm. The reaction chamber cross section is preferably circular, pear shaped, hexahedral, octahedral and / or rectangular in cross section. The reaction chamber preferably has a substantially circular inlet capillary that runs perpendicular to the bottom region, and its radius preferably has from about 5 μm to about 50 μm, in particular from about 10 μm to about 20 μm. A sharp-angle inlet capillary appears to be less suitable because its sharp angle acts like a capillary plug and at least slows (or completely stops) fluid flow. It is advantageous if the reaction chamber preferably has a recess leading to at least one vent located opposite the inlet capillary muscle.
In a particularly preferred embodiment, the reaction chamber has a substantially hydrophobic design in the upper region that is coplanar with the hydrophobic portion of the inlet channel, while the reaction chamber is under the hydrophobic region of the inlet channel. The lower region has a substantially hydrophilic design. As a result, of course, in all embodiments of the invention, the distributor and the inlet channel can complement each other, i.e. the sample holder has both at least one distributor channel and at least one inlet channel, or The function of the distributor or inlet channel is usually indicated by at least one channel, ie the sample holder has either at least one distributor channel or at least one inlet channel. The function of the channel can thus be taken up by the distributor channel or newly supplemented. Furthermore, the invention includes any desired combination between the reaction chamber, inlet channel and / or distributor channel. As described above, according to the present invention, the lower region of the reaction chamber is provided with a hydrophilic design, particularly preferably in a form in which the hydrophilization increases in layers. However, it is necessary under certain conditions that the lower part of the reaction chamber is at least partially hydrophobic (similar) design. This is advantageous, for example, when it results in a strong reverse capillaryization in the case of a hydrophilic surface to which a dry solution containing a surfactant is added to improve the solubility of the sample material. In order to avoid this effect, the reaction chamber usually has a hydrophobic design which is an outcome of the invention. The surfactant then forms a hydrophilic film on the hydrophobic surface by solution drying. The reaction chamber preferably has at least one round corner. All corners of the reaction chamber (other than the corner with the inlet capillary) may be rounded again. Capillary forces are strongly suppressed with this design of the reaction chamber corner which again greatly improves the injection kinetics (radius ≧ 100 μm). Furthermore, the invention provides that the reaction chamber sidewalls have a substantially smooth and / or corrugated design. In this case, simultaneous expansion of the surface due to the corrugated structure occurs, but the sidewalls of the corrugated design (radius preferably about 30-50 μm) can act as vertical capillaries. This arrangement allows the solution to be quickly and evenly distributed over a relatively large surface so that the introduction of sample material into the solution accelerates the drying process with the “release” of the inlet capillary. Re-dissolution upon addition of sample material is also improved. The corrugated structure of the side walls may extend over various regions of the wall. As a result, for example, there is no corrugated structure near the vent structure, but it may extend from the bottom to the cover near the inlet capillary. In such a corrugated structure distribution, it is proved that the cover element is wetted by the fluid flowing into the inlet capillary and the corrugated structure area that follows, and that the delay effect of the remaining part is strong enough to allow time for air to escape. It was. The jagged structure seems disadvantageous because it prevents the bottom from getting wet and cannot be led to the bottom.
In a further advantageous result of the invention, it is provided that the sample holder is covered in a fluid tight manner with a cover element. In addition to the appropriate shape of the capillary as described above, the sample holder should be sufficiently well sealed (if appropriate, the sample material and / or the sample material to obtain sufficient capillary force to passively transport the sample fluid to the microfluidic sample holder. It is also important to do (after reagent introduction). The cover element is preferably a membrane with one side with a suitably thick adhesive layer. The membrane and / or adhesive is preferably a heat activatable and / or pressure sensitive membrane or adhesive. In the past, strong hydrophobic adhesives (e.g., silicone adhesives, rubber adhesives or silicone rubber adhesives) have been used for non-surface treated plastics (polystyrene, polypropylene, Polycarbonate, polymethylmethacrylate), which is still more hydrophobic, has been postulated to disturb the fluidics of capillaries. It is an achievement of the present invention to show that these adhesives are particularly suitable for sealing cover elements. As a result, in a particularly preferred embodiment, when a fluoropolymer membrane is used as the membrane, the uncoated surface that prevents the sample holder becomes very hydrophobic, has good slipperiness, and is very strong which is advantageous for optical measurement methods Resistant to contamination. The membrane is applied by a roll, preferably under pressure, preferably about 2 to 5 bar, so that the sample holder is substantially gapless. The adhesive is preferably an adhesive. Adhesives have the property of avoiding “free space” under pressure. This is used in daily life for the purpose of showing gaps, for example. In the case of sealing (the pressure is not too high and the adhesive layer is not too thick), this effect is advantageously used to suppress unwanted capillary forces between the side wall and the cover. It has been found that when the sample holder is sealed, “microbeads” are formed in-situ and prevent this effect (capillization between the side wall and the cover) as well as its hydrophobicity. Furthermore, the adhesive layer wets only with a delay, and when the bubbles are trapped in the test recess (sample receiving chamber, reaction chamber, etc.) when injected into the test recess (eg, sample receiving chamber, reaction chamber, etc.), Before reaching the structure, there is sufficient time for the air to escape from the vent structure opposite the injection side. Hydrophobic adhesives have proven to be particularly suitable adhesives. This adhesive is, for example, the aforementioned silicon adhesive, rubber adhesive, silicon rubber adhesive and / or fluoropolymer adhesive.
In a further preferred embodiment of the invention, the sample holder according to the invention is used in microbiological diagnostics, immunology, PCR (polymerase chain reaction) methods, clinical chemistry, microanalysis and / or active substance testing.
The invention further provides a method for analyzing at least one sample material when added to a sample holder according to the invention, having at least one surfactant to which a sample medium is added. The surfactant is preferably a nonionic surfactant. The nonionic surfactant is preferably a material having an HLB (Hydrophilic Lipophilic Balance) number between about 9 and about 13. The surfactant is preferably a propylene oxide / ethylene oxide triblock polymer, alkyl polyglycoside, nonylphenyl ethoxylate, ethoxylate of secondary alcohol, octylphenyl ethoxylate, polyethylene lauryl ether and / or sorbitan ester. Additional examples of nonionic surfactants are well known to those skilled in the art and can be collected from the appropriate specialist literature. Examples of surfactants in this group include:
In the group of propylene oxide / ethylene oxide triblock polymers, Pluronic 10300 (manufactured by BASF)
In the group of alkylpolyglycosides, Glucopone 650 (manufactured by Cognis)
Nonylphenyl ethoxylate group has Tergitol NP7 and Tergitol NP9 (Dow Chemical)
Tergitol 15S7 and Tergitol 15S9 (Dow Chemical Co.) in the ethoxylate group of secondary alcohols
In the octylphenyl ethoxylate group, Triton X45 and Triton X114 (Dow Chemical Co.)
Bridge 30 in the polyethylene lauryl ether group
Tween 20 in the sorbitan ester group
In addition to a variety of new structural elements for the design of diagnostic microfluidic sample holders, the general three-dimensional design of this sample holder for various functional levels of hydrophilization is described in the invention filed here. Finally, a free surface energy gradient is proposed to optimize fluidics and plug function. Since the distributor and / or inlet channels are not wetted by the additive-free aqueous medium, they are partially non-capillary at the bottom, but are largely contradicting that they are predominantly mainly hydrophobic designs. Sounds like But this is intentional. When a low concentration of a suitable surfactant is added to the sample medium as described above, which fluid has sufficient free surface energy to wet the hydrophobic structure. The non-ionic surfactants described are of little toxicity and are therefore mainly considered for diagnostic purposes. As mentioned above, nonionic surfactants are used as additives in many diagnostic and biotechnological methods, but as emulsifiers and solubilizers for pharmaceuticals, or even wetting agents in detergents, cleaning agents, coloring media, etc. As mainly spread. These chemically very different materials have mostly asymmetric designs, i.e., for example, a hydrophilic head and a hydrophobic tail. However, there are also symmetrically designed copolymers (ethylene oxide (EO) / propylene oxide (PO) compounds) with a hydrophobic core and hydrophilic ends. However, not all surfactants have excellent wettability. These are substantially all substances used as emulsifiers (low HLB number = hydrophilic / hydrophobic balance) or solubilizers (high HLB number). A good wetting agent is a substance with an HLB number between 9 and 13, such as the surfactants described above. Some of these materials are unsuitable due to highly foamed compounds. Compounds which have an optimum wetting effect at the lowest possible concentration and which have little or no foaming are suitable (see substance above). One property of a good wetting agent emerges from the solution at the interface between the fluid and the solid surface and is absorbed by that surface. As a result, the concentration in the fluid decreases in proportion to the wetting surface until the critical limit is reached. Pluronic 10300 manufactured by BASF is an aqueous medium having a Pluronic 10300 concentration of 0.03% and can provide sufficient free surface energy to wet the distributor flow path. In this case, the fluid first flows through the flow path much more slowly than in the fully hydrophilic design structure. The fluid then strikes the interface between the hydrophobic upper structure and the hydrophilic lower structure at the inlet (in the capillary) to the sample recess (eg, reaction chamber). Downward capillary action is preferred not only for vertical capillaries but also for energy requirements. This fluid quickly reaches the bottom, wets the bottom and quickly rises in the test recess (eg, reaction chamber) until it reaches the hydrophobic layer (first layer near the inlet side / inlet capillary). In this case, wetting of the remaining surface is delayed. The wetting of the sealing layer takes place at the inlet side / inlet capillary in the direction of the ventilating capillary slowly so that all air can escape. Liquids containing only low concentrations of surfactant will stop at a completely hydrophobic vent structure, combining structural elements and energy unfavorable conditions. If the sealing layer is initially hydrophobic, i.e. more hydrophobic than the sample holder, certain other substances (Tergitol NP9) can be injected into the untreated, i.e. hydrophobic polystyrene sample holder without failure. If the sealing layer is more hydrophilic (eg, acrylate adhesive), the fluid will capillary along the side between the sample holder and the sealing layer. Test recesses (eg reaction chambers) cannot be injected.
Finally, the invention provides a kit for microbiological diagnostics, immunology, PCR (polymerase chain reaction) method, clinical chemistry, microanalysis and / or active substance testing comprising a sample holder according to the invention.
Further details and characteristics of the invention are given in the following description of preferred exemplary embodiments in connection with the subclaims. Here, each characteristic can be implemented by itself or separately. The invention is not limited to this exemplary embodiment.
Exemplary embodiments are shown schematically in the figures. In this case, the same reference numerals in the drawings designate the same elements, elements having the same function, or functions corresponding to each other.
Multiplications and developments of the exemplary embodiments described can be made within the scope of the invention.
The specified range includes all sub-intervals that are all considered to be intermediate values even if they are not named.
FIG. 1 shows a schematic plan view of the sample holder (10). Various modifications of this structure can be considered independently in the reaction to be carried out (left half and right half of the sample holder). Similarly, each structure is formed in a different shape depending on the necessity in each case. Specifically, the sample receiving chamber (12), the distributor channel (14) extending from the sample receiving chamber (12), and the left of the sample holder (10) in which the sample receiving chamber (12) is directly connected to the reaction chamber (16). While the half distributor channel (14) is present, the right half of the sample holder (10) is provided with an inlet channel (18) branched from the distributor channel (14) and a sample receiving chamber (12) and a reaction chamber ( 16) between. Vent structures or vent capillaries each open to the vent (20) branch off in these reaction chambers (16). The sample holder (10) shown in FIG. 1 consists of a basic structure of a microfluidic sample holder that does not show inventive additional structures that are at least partially of a hydrophobic design. These additional structures are illustrated in the following figures.
FIG. 2 shows a schematic perspective side view of the sample holder (10) according to FIG. Various designs of the sample receiving chamber (12), the distributor channel (14) branched therefrom, as well as the reaction chamber (16) and the vent (20) can be seen again. The inlet channel (18) branches off at the distributor channel (14) in the right half of the sample holder (10).
FIG. 3 shows a schematic diagram of microbead production at the transition between the sidewall and the cover element. The provision of the cover element (40) on one side of the adhesive layer has an advantageous effect when the sample holder is sealed with the cover element. When this adhesive layer is an adhesive, an undesirable capillary force between the side wall and the cover can be suppressed. At this location, i.e. between the side wall and the cover, "microbeads" are formed (denoted by the arrows in Fig. 3) to ensure that capillarization between the side wall and the cover element is suppressed.
FIG. 4 shows a schematic view of an advantageous embodiment of the sample holder. In this case, an additional structure (22) arranged between the reaction chamber (16) and the vent (20) is shown in the figure. The additional structure (22) in FIG. 4 is a semi-circular recess (24) positioned opposite the distributor channel (14). An additional capillary (28) designed in the acute angle element (28) of FIG. 4 follows this semicircular recess (24).
An advantageous improvement (22) of the additional structure is shown in FIGS. 5A-5C. Here, FIG. 5A shows a zigzag design structure, while FIG. 5B shows a structure showing a steep angle of 90 degrees or more. FIG. 5C shows an element with an acute angle element (28) of varying structure depth, followed by an additional capillary (30) that leads directly or through the adjacent structure to a terminal recess with a valve function. ing.
FIG. 6 shows a schematic view of an advantageous embodiment of a sample holder for performing a continuous analysis. Distributor channel (14), two reaction chambers (16) of different sizes, semi-circular recess (24) additional structure (22) design, and semi-circular recess (24) in vent (20) A connected vent capillary is shown. In this arrangement, the first reaction chamber (16), which is the larger reaction chamber in FIG. 6, is injected as soon as the sample is arranged therein, and the first-stage reaction proceeds. This is because only the larger reaction chamber (16) is injected by the open vent (20) on the side of the first reaction chamber (16). Only when the venting system of the second reaction chamber (not shown in FIG. 6) is opened, the sample can flow from the larger reaction chamber (16) into the smaller second reaction chamber (16). The second stage reaction continues in it.
7A-7C show an advantageous arrangement of sample holders. FIG. 7A shows a sample holder in a “jellyfish-like” arrangement, where the jellyfish-like head attempts to represent the sample receiving chamber (12), while the “tentacle” functions as a distributor channel and / or inlet channel. Undertake. 7A shows the reaction chamber (16) and the vent (20). FIGS. 7B and 7C further show possible improvements of the sample holder according to the invention, in which the sample receiving chamber (12) is again formed in the center of a circular (FIG. 7C) or elongated structure (FIG. 7B) The channel and / or inlet channel (14/18) begins there. A reactant (16) and a vent (20) are disposed around the sample receiving quality (12).
8A-8D show an advantageous improvement of the reaction chamber. Here, the cross section of the reaction chamber (16) is circular (FIG. 8A), pear-shaped (FIG. 8B), hexahedral (FIG. 8C) or rectangular (FIG. 8D). Further shown is an inlet capillary (36) and a recess (38) located opposite the muscle of the inlet capillary (36).
FIG. 9 shows a schematic view of the reaction chamber side wall. The corrugated side walls of the corrugated structure that act as vertical capillaries with the corrugated structure as the surface expands are shown. When the sample material is introduced into the solution by this arrangement, the sample material is quickly and uniformly distributed over a relatively large surface to facilitate the drying process while simultaneously “releasing” the inlet capillary.
FIG. 10 shows a schematic view of the reaction chamber side wall width. Sidewall corrugations can extend to various regions. FIG. 10 shows the corrugated structure extending from the bottom to the cover near the inlet capillary, but not near the vent structure. The distribution of this corrugated structure proves that the cover element is wetted by the inlet capillary region and the subsequent fluid flowing into the corrugated structure, and that the regeneration effect at the remaining part is strong enough to allow air to escape. .
One-step assay
A simple design consisting of a sample receiving chamber, a distributor channel and / or an inlet channel, a reaction chamber and a vent (including the supply structure) is a (multi-parameter) one-step assay (antigen detection, microbiology) Sufficient for testing). In this case, the end “vent valve” or vent is open (see FIG. 11).
One-step assay method-antibody testing
First, if the vent remains closed in the case of the sample holder as in FIG. 11, the first reaction stage can be performed at the sample receiving site (12). This is demonstrated in the examples, for example by a simple antibody test for the detection of pathogens of airway diseases. Magnetic particles coated with anti-human immunoglobulin (Ig) A or human antibody IgM on one (left) side of the reaction chamber (16), as well as fluorescently labeled antigens (eg Rous sarcoma virus (RSV), influenza, etc.) On the other hand, only the labeled antigen is arranged on the right side. Paramagnetic nanoparticles coated with anti-human IgG in serum samples diluted 1:10 to 1:50 at a concentration sufficient to bind all IgG are placed in the sample receiving chamber (12). Once this first incubation step is complete, a strong magnetic field is applied to the sample receiving chamber (12) and the left vent (20) is opened. The sample immediately flows into the reaction chamber (16), and each of IgA and IgM binds to the magnetic particles. When certain IgM or IgA are present, they bind to an appropriately labeled antigen. This reaction is evaluated with a three-dimensional (3D) fluorescence scanning system or other optical detection system. Once the left reaction chamber (16) is full, the magnetic field is turned off and the right vent (2) is opened. Samples with nanoparticles immediately flow into the right reaction chamber (16) and specific IgG antibodies can be detected as well after an additional incubation step. This method is also suitable for the determination of IgG subnets or appropriately modified IgE, for example allergy determination.
Two-step assay
(With or without one-step assay)
FIG. 12 shows a design for performing a two-step assay. This design can also provide a structure for performing a one-step assay at the same time. Closing all valve functions (ie all vents) allows the sample to be adjusted where appropriate. Two-step assays are well known in clinical chemistry, for example, assuming that the first reaction step is an enzymatic reaction that detects the final product with the aid of reagents that are inconsistent with the enzymatic reaction. Another example is a reaction in coagulation diagnosis in which only the extra specimen in the sample is detected, i.e. a specific defined fraction of the specimen needs to be inactivated in the first stage regardless of type. In these assays, the first stage chamber is approximately five times the second stage test recess that does not begin until the end vent is opened. The difference in size ensures that only the material for which the first stage has been performed reaches the second chamber.
Special case: Antibody detection
If the sample holder is coated with antibody and antigen and must be used, for example, to detect beads placed in the first chamber, the first chamber is much smaller than the second chamber. The second chamber is then used only as a “garbage bin” for the sample and the wash. These steps can be easily controlled by opening and closing the terminal vent.
Special case: PCR sample holder
FIG. 13 shows a special case of the PCR sample holder. This sample holder is capable of performing PCR when appropriate, or, if appropriate, DAN / RNA isolation and subsequent target detection after the second specific PCR. The sample holder is about 2-3 mm thick and has a hydrophobic design with a thickness of 50-100 μm up to the middle layer. The bottom of the sample holder consists of a plastic-coated metal thin film front (IA) and a heat-resistant plastic back. The cover is a highly elastic film coated with an adhesive. The sample receiving chamber (12) (20-100 μl) is used for sample preparation. The recess (12) can accommodate all the reagents necessary for isolation. Subsequent materials that should not be transferred to the process bind to the solid phase (eg, magnetic particles). Once isolation is complete, the vent (20 ') is opened and the vent (20) is mechanically closed while the sample (enhanced by heating if appropriate) passes through the distributor channel (14). It flows into the reaction chamber (16). All reagents for PCR are partly bound to the solid phase and placed in the reaction chamber (16) if necessary to optimize this method. After performing PCR (multiple or specific), the vent (20 ") is opened and the amplified product passes through the channel system to the reaction or detection chamber (16 '). The distributor channel (14) is initially twisted. The shape is completely hydrophobic, then narrows but deepens, then goes down into a hydrophilic layer, and the narrower inlet channel (18) is likewise hydrophobic at the bottom. The tortuous structure of the flow path (14) and the closed valve or closed inlet structure (20 ") prevent premature transfer from the reaction chamber (16) to the detection chamber (16 '). During PCR, the inlets (20) and (20 ') are blocked from the outside. The inlet capillary is connected separately to the inlet (20 ") and includes a capillary plug structure as previously described. As an option, a second PCR can be performed or its detection can have various shapes. This can be done directly in the chamber (16 '), so traps and detection probes (eg hairpins) can then be attached to the beads, and we are not going to go into the details of the variety of variants here.

FIG. 1 shows a schematic plan view of a sample holder. FIG. 2 shows a schematic perspective side view of the sample holder. FIG. 3 shows a schematic diagram of microbead production at the transition between the sidewall and the cover element. FIG. 4 shows a schematic view of an advantageous embodiment of the sample holder. Figures 5A-5C show an advantageous improvement of the additional structure. FIG. 6 shows a schematic view of an advantageous embodiment of a sample holder for performing a continuous analysis. Figures 7A-7C show an advantageous arrangement of sample holders. Figures 8A-8D show an advantageous improvement of the reaction chamber. FIG. 9 shows a schematic view of the reaction chamber sidewall. FIG. 10 shows a schematic view of the reaction chamber side wall width. FIG. 11 shows a schematic diagram of an advantageous arrangement of a sample holder for a (multi-parameter) one-step assay. FIG. 12 shows a schematic diagram of an advantageous arrangement of a sample holder for a two-step assay. FIG. 13 shows a schematic view of an advantageous arrangement of the PCR sample holder.

Explanation of symbols

10 Sample holder 12 Sample receiving chamber 14 Distributor channel 16 Reaction chamber 18 Inlet channel 20 Vent 22 Additional structure 24 Semicircular recess 26 Capillary 28 Sharp angle element 30 Additional capillary 36 Inlet capillary 38 Recess 40 Cover element

Claims (31)

A sample holder (10)-at least one sample receiving chamber (12) for the sample fluid;
At least one distributor channel (14) extending from each sample receiving chamber (12) with at least one distributor channel (14) connected to at least one sample receiving chamber (12);
- less and at least one reaction chamber injection channel which branches one of the distributor passages (14) (18) is opened even (16), and - at least one vent opening for each reaction chamber (16) Part (20)
A sample holder (10) between the sample receiving chamber (12), the distributor channel (14), the reaction chamber (16), the injection channel (18) and / or the vent opening (20). Having at least one additional structure (22) that is at least partly a hydrophobic design;
The upper region of the distributor channel (14) and / or the injection channel (18) is in the plane of the vent opening (20) and is of a hydrophobic design, while the distributor is below the vent opening (20) The lower region of the channel (14) and / or the injection channel (18) is a hydrophilic design,
The upper region of the reaction chamber (16) is in the hydrophobic part plane of the distributor channel (14) and / or the injection channel (18) and has a hydrophobic design, while the lower region of the reaction chamber (16) Is in the lower region below the distributor channel (14) and / or the injection channel (18) and has a hydrophilic design, or the reaction chamber (16) has a feature that is a fully hydrophobic design . In the case of a reaction chamber (16) with a hydrophilic design, the sample holder according to claim 1, characterized in that the hydrophilization increases in layers in the lower region of the reaction chamber (16). Sample holder according to claim 1 in cross-section is from about 10μm to about 300μm of each additional structure (22). 2. Sample holder according to claim 1, characterized in that the additional structure (22) is a semicircular recess (24) arranged opposite the distributor channel (14). 5. Sample holder according to claim 4, characterized in that at least one additional capillary (26) extends from the semicircular recess (24) and the additional capillary (26) is characterized by an acute-angled or zigzag-shaped design. 6. Sample holder according to claim 5, characterized in that the extension from the additional capillary (26) is at least one additional element (28) with an acute angle and varying structural depth. At least one additional capillary (30) extends away from the element (28) having an acute angle and a change in structure depth, and the additional capillary (30) opens directly into the terminal recess having a valve function or opens through an adjacent structure. The sample holder as claimed in claim 6.   2. The sample holder according to claim 1, wherein the distributor channel (14) connected to the sample receiving chamber (12) has a serpentine design feature. Multiple vent openings (20), distributor channels (14), injection channels (18) , reaction chambers (16) and / or additional structures (22) around or parallel to the sample receiving chamber (12) 2. A sample holder according to claim 1 having arranged features. The sample holder of claim 1, wherein the reaction chamber (16) is characterized by a vertical length of about 500 µm to about 3 mm. Sample holder according to claim 10, the edge length of the reaction chamber (16) has a characteristic of about 300μm is about 1mm on average. The reaction chamber (16) a circular cross section, pear, hexahedral, octahedral and / or rectangular design sample holder according to claim 10 or 11 having the features of. 13. Sample holder according to one of claims 10 to 12, characterized in that the reaction chamber (16) has a circular injection capillary (36) running perpendicular to the bottom region. Sample holder according to claim 13, injection capillary (36) radius has a characteristic about 5μm about 50 [mu] m. 15. Sample holder according to one of claims 10 to 14, characterized in that the reaction chamber (16) is arranged opposite the injection capillary (36) muscle and has a recess (38) following at least one vent opening (20).   17. Sample holder according to claim 12, characterized in that the reaction chamber (16) has at least one rounded corner. 16. Sample holder according to claims 10 to 15, characterized in that the reaction chamber (16) has smooth and / or corrugated side walls.   2. Sample holder according to claim 1, characterized in that the sample holder (10) is covered in a fluid tight manner by a covering element (40).   19. Sample holder according to claim 18, characterized in that the covering element (40) is a film with an adhesive layer of suitable thickness on one side.   20. Sample holder according to claim 19, characterized in that the film and / or adhesive is a heat activated and / or pressure sensitive film or adhesive. 21. Sample holder according to claim 19 or 20, characterized in that the sample holder (10) is pressed against the film with a roll so as to cover it gaplessly .   A sample holder as claimed in one of claims 19 to 21, characterized in that the film is a fluoropolymer film.   21. A sample holder according to claim 20, wherein the adhesive is a tacky adhesive. Sample holder according to claim 23 having the features adhesive is a hydrophobic adhesive.   25. Sample holder according to claim 24, characterized in that the adhesive is silicon, rubber, silicone rubber and / or fluoropolymer adhesive.   Use of the sample holder according to claim 1 in microbiological diagnostics, immunology, PCR (polymerase chain reaction), clinical chemistry, microanalysis and / or active substance testing.   A sample material analysis method for analyzing a sample material, wherein the sample medium is applied to a sample holder as claimed in one of claims 1 to 25, to which at least one surfactant is added.   28. The method of claim 27, wherein the surfactant is a nonionic surfactant.   29. The method of claim 28, wherein the nonionic surfactant is a substance that is between about 9 and about 13 hydrophilic and hydrophobic balance (HLB).   30. The surfactant is characterized in that it is a propylene oxide / ethylene oxide triblock polymer, alkyl polyglycoside, nonylphenyl ethoxylate, secondary alcohol ethoxylate, octylphenyl ethoxylate, polyethylene lauryl ether and / or sorbitan ester. How to charge.   A kit for microbiological diagnostics, immunology, PCR (polymerase chain reaction), clinical chemistry, microanalysis and / or active substance testing comprising the sample holder as claimed in one of claims 1 to 25.

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